With the help of ground squirrel hepatitis virus and woodchuck hepatitis virus as models for hepatitis B virus, we have identified the major replicative DNA intermediates and identified the major signals controlling minus and plus strand DNA synthesis. Furthermore, we have developed a novel system allowing for the synchronized replication of duck hepatitis B virus in cultured cells, employing phosphonoformate, a reversible inhibitor of the hepadnavirus reverse transcriptase. This system permits accumulation and subsequent isolation of DNA replicative intermediates at different stages of maturation and therefore, allows for an examination of several aspects of the hepadnavirus replication cycle that have previously not been accessible for a biochemical analysis. For this proposal we have focused on four aims, some of which have directly emerged from our previous genetic and biochemical studies on viral DNA synthesis supported by the FIRST award. We will investigate the physical structure and arrangement of pregenomic RNA in core particles. These studies will for the first time shed light on the earliest events of the viral replication cycle where pregenomic RNA, polymerase and core protein assemble into an initiation complex to begin viral DNA synthesis. In addition, we will examine the biochemical conditions under which RNA containing core particles synthesize DNA in vitro and use a new strategy for the expression of enzymatically active reverse transcriptase. We will also continue ongoing efforts aimed at an understanding of the mechanism by which plus strand DNA synthesis is primed and perform a biochemical and structural investigation of core particles, in which DNA synthesis has been arrested at a step when plus strand priming occurs. We will determine whether priming of plus strand DNA synthesis depends on a precise spatial arrangement of minus stand DNA in core particles. Finally, we will present an experimental strategy to determine at what stage during DNA synthesis cytoplasmic core particles assemble with the viral envelope components and what the signals are that control this reaction. The information gained from these investigations will not only help to complete our understanding of the mechanism of hepadnavirus replication but also reveal new target sites for the design of effective antiviral compounds required for the treatment over more than 200 million individuals chronically infected with hepatitis B virus.